Bibliographic details of the publications referred to by author in this specification are collected alphabetically at the end of the description.
Reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that this prior art forms part of the common general knowledge in any country.
Interleukin-12 (also known as cytotoxic lymphocyte maturation factor or natural killer cell stimulatory factor) is a 75-kDa heterodimeric protein. It consists of a p35 subunit which is comprised of a bundle of four alpha helicies that resembles the class I cytokines. The 11 amino acids C-terminal to C63 in p35 are termed the disulphide bond loop, as this region contains numerous residues that contact p40, the other subunit of IL-12, including an interchain disulphide bond with C177 in p40. The p40 subunit folds like the extracellular domain of other class I receptors, such as growth hormone receptor (GHR).
The p40 subunit has three domains labelled D1, D2 and D3. Each domain is a β-sheet structure with the D2 domain containing the C177 interchain disulphide bond. There is also an N-linked glycosylation site (GlcNAc-GlcNAc-mannose, where GlcNAc is N-acetylglucosamine) on D2 (Yoon et al. 2000 Embo J 19 3530-41).
Interleukin-23 was discovered more recently (2000) by searching sequence databases with a computationally derived profile of members of the interleukin-6 helical cytokine family. This search led to the discovery of a novel cytokine subunit which was named IL-23p19 (p19). This subunit was co-expressed with IL-12p40 leading to secretion of a heterodimeric protein called Interleukin-23 (IL-23). IL-23p19 is thought to resemble IL-12p35 in that it contains a four helix bundle (Oppmann et al. 2000 Immunity 13 715-25).
The specific effects of IL-12 on its target cell types are mediated by the IL-12R complex, which consists of IL-12Rβ1 (Chua et al. 1994 J Immunol 153 128-36) and IL-12Rβ2 (Presky et al. 1996 Proc Natl Acad Sci USA 93 14002-7). The specific effects of IL-23 on its target cell types are meditated by the IL-23R complex, which consists of IL-12Rβ1 and IL-23R (Parham et al. 2002 J Immunol 168 5699-708).
IL-12Rβ1 binding to IL-12 and IL-23 is mediated via the common p40 subunit. This was demonstrated using competition experiments in which a homodimer of the p40 subunit (p80) competed with IL-12 for binding to IL-12Rβ1. However p80 had no effect on the binding of IL-12 to IL-12Rβ2 (Presky et al. 1996 Ann NY Acad Sci 795 390-3).
It follows that the p35 subunit or the heterodimeric interface of IL-12 is responsible for binding to IL-12Rβ2 thereby conferring IL-12 selectivity on the IL-12R complex (Trinchieri et al. 2003 Immunity 19 641-4). Likewise p19 or the heterodimeric interface of IL-23 is responsible for binding to IL-23R thereby conferring IL-23 selectivity on the IL-23R complex.
The signalling of the IL-12R and IL-23R complex has been elucidated. The IL-12R activates the Janus kinase (JAK)—signal transducer and activator of transcription (STAT) pathway of signal transduction. The actual cellular effects of IL-12 are due mainly to STAT4 activation (Kaplan et al. 1996 Nature 382 174-7). There is a STAT4 binding site on IL-12Rβ2 indicating that this receptor is vital for signalling (Yao et al. 1999 Arch Biochem Biophys 368 147-55). This is also demonstrated in the correlation of the expression of IL-12Rβ2 to the responsiveness of TH1 cells to IL-12 (Rogge et al. 1997 J Exp Med 185 825-31). The IL-23R activates similar complexes to IL-12R such as the JAK-STAT pathways. STAT3 is more prominently induced than STAT4 by binding of IL-23 to the IL-23R and the other resulting DNA-binding STAT transcription factor complexes are different (Parham, Chirica et al. 2002 J Immunol 168 5699-708).
The biological effects of both IL-12 and IL-23 are distinct from each other. IL-12 is secreted by activated inflammatory cells (monocytes, macrophages, neutrophils, microglia, dendritic cells). IL-12 has mainly been studied for its effects on lymphocytes, although it affects other types of cells also. During the inflammatory response, IL-12 induces NK cells and T cells to produce interferon-γ (IFN-γ). Then IL-12, possibly in combination with IFN-γ, induces T cells to differentiate into TH1 cells. This response stimulates the cellular immune system and maximises the killing effect of macrophages on pathogens and the proliferation of CD8+ T cells (Trinchieri 2003 Nat Rev Immunol 3 133-46). Overproduction of IL-12 has been correlated with heightened proinflammatory activities and tissue damage typical of autoimmunity (Leonard et al. 1997 Crit. Rev Immunol 17 545-53). Dysregulated IL-12 production has been implicated in the following diseases: psoriasis (Yawalkar et al. 1998 J Invest Dermatol 111 1053-7; de Rie 1999 Dermatology 199 101; Shaker et al. 2006 Clin Biochem 39 119-25), Crohn's Disease (Neurath et al. 1995 J Exp Med 182 1281-90; Simpson et al. 1998 J Exp Med 187 1225-34; Camoglio et al. 2002 Eur J Immunol 32 261-9), Multiple Sclerosis (Fassbender et al. 1998 Neurology 51 753-8; Laman et al. 1998 J Neuroimmunol 86 30-45), rheumatoid arthritis (Kim et al. 2000 Clin Exp Immunol 119 175-81; Leung et al. 2000 J Immunol 164 6495-502) among other autoimmune diseases. The role of IL-12 in these diseases is not clear however it is thought that the overpolarisation of the TH1 response may be involved (Gordon et al. 2005 Curr Opin Gastroenterol 21 431-7).
IL-23 is secreted by activated human macrophages as well as dendritic cells (Verreck et al. 2004 Proc Natl Acad Sci USA 101 4560-5). IL-23 predominantly acts on memory T-cells and has been postulated to promote autoimmune disease through the regulation of IL-17A and IL-17F as demonstrated in the ability of murine splenocytes to secrete IL-17 in response to IL-23. In humans the IL-23/IL-17 pathway is present, and IL-23 has been shown to be an equally good inducer of IL-21, IL-22, IFN-γ, TNF-α along with IL-17, all pro-inflammatory cytokines. In vitro IL-6 and TGF-β1 promote naïve T-cells down a newly discovered T-cell pathway (TH17) (Zhou et al. 2007 Nat Immunol 8 967-74). These cell are further driven in a autocrine manner via secretion of IL-21. Lastly, IL-23 and/or IL-1β are thought to maintain cells in this TH17 response (For a review see Dong 2008 Nat Rev Immunol 8 337-48). Also of interest is the transcription factor RORγt which has been shown to be upregulated in the TH17 response (Chen et al. 2007 Arthritis Rheum 56 2936-46).
Since both IL-12 and IL-23 contain a common subunit, it has been difficult to attribute disease states solely to overproduction of one interleukin or the other. However research indicates that IL-23 dysregulation has been implicated in the following diseases: psoriasis (Lee et al. 2004 J Exp Med 199 125-30; Torti et al. 2007 J Am Acad Dermatol 57 1059-68), Crohn's disease (Neurath 2007 Nat Med 13 26-8) and Multiple Sclerosis (Cua et al. 2003 Nature 421 744-8) among other autoimmune diseases.
IL-12p40 can be secreted as a monomer (IL-12p40) or as a homodimer (IL-12p80) which is two p40 subunits held together by a disulphide bond (Gillessen et al. 1995 Eur J Immunol 25 200-6). These p40 species are secreted at 50-fold excess compared with IL-12 in a murine shock model (Gillessen, Carvajal et al. 1995 Eur J Immunol 25 200-6) and 10-20 fold excess in human peripheral blood mononuclear cells (PBMCs) (D'Andrea et al. 1992 J Exp Med 176 1387-98). IL-12p80 can antagonise IL-12 activity in vitro 20-fold greater than that of IL-12p40 (Gillessen, Carvajal et al. 1995 Eur J Immunol 25 200-6). Recombinant IL-12p80 has been shown to bind to IL-12β1 (Wang et al. 1999 Eur J Immunol 29 2007-13).
IL-12p40/p80 is considered an antagonist of the IL-12/23 receptor complex because recombinant murine IL-12p80 (rmIL-12p80) has been shown to compete with IL-12/23 binding to IL-12Rβ1 in vivo and in vitro (Mattner et al. 1993 Eur J Immunol 23 2202-8; Gillessen, Carvajal et al. 1995 Eur J Immunol 25 200-6; Gately et al. 1996 Ann NY Acad Sci 795 1-12). The homodimer has also been shown to prevent IL-12 mediated shock in the murine model (Mattner et al. 1997 Infect Immun 65 4734-7). In an investigation of IL-23 mediated immunological functions IL-12p40 impaired IL-23 induced cytokine production by competitive binding to the IL-12Rβ1 (Shimozato et al. 2006 Immunology 117 22-8). More recently IL-12p40 or IL-12p80 has been implicated in other biological roles. One of the earliest established activities of IL-12p80 is as a chemoattractant for macrophages. IL-12Rβ1 deficient macrophages but not IL-12Rβ2 or IL-12p35 deficient macrophages, have reduced chemoattractive responses to rmIL-12p80, indicating that IL-12Rβ1 can mediate responses to IL-12p80 in the absence of IL-12Rβ2 and that IL-12p80 activity is independent of IL-12 (Russell et al. 2003 J Immunol 171 6866-74). IL-12p80 can also act as an inducer of dendritic cell (DC) migration. IL-12p40-deficient DCs are unable to migrate from the lungs to the lymph nodes in response to mycobacteria. The fact that the loss of both IL-12p35 and IL-23p19 did not impact on the ability of mycobacterially activated DCs both to migrate in response to chemokines and to drive T-cell expansion highlights this unique role for IL-12p40 (Khader et al. 2006 J Exp Med 203 1805-15). IL-12p40 and IL-12p80 have also been shown to mediate inflammatory responses in the lung and has been shown to induce IFN-γ production by CD8+ T cells (Cooper et al. 2007 Trends Immunol 28 33-8).
Since IL-12 and IL-23 have been implicated in a variety of disorders several therapeutic strategies have been designed to inhibit IL-12 and/or IL-23 activity. Some of the earliest described antibodies were murine monoclonal antibodies that were secreted by hybridomas of mice immunised with IL-12 (Strober et al. PCT Publication No. WO 97/15327; Gately et al. WO99/37682 A2, Neurath, Fuss et al. 1995 J Exp Med 182 1281-90). The use of these murine antibodies for treating humans is limited due to issues arising from administration of a mouse immunoglobulin to humans. Such issues include the raising of auto-antibodies against the mouse immunoglobulin thereby removing its presence in the serum and negating any therapeutic effect. This effect known as the human anti-mouse antibody (HAMA) was overcome in part with the advent of chimeric antibodies which limited the murine sequence to only the variable regions of the antibody (Junghans et al. 1990 Cancer Res 50 1495-502; Brown et al. 1991 Proc Natl Acad Sci USA 88 2663-7). Chimeric antibodies have been described that bind to IL-12 (Perritt et al. PCT publication No. WO 02/097048A2). Even more human-like antibodies are ‘humanised’ antibodies' which contain the complementarity determining regions of a donor murine antibody but have variable framework regions derived from a human acceptor antibody (Jones et al. 1986 Nature 321 522-5, Winter U.S. Pat. No. 5,225,539, Queen et al. U.S. Pat. No. 5,693,761). Such ‘humanised’ antibodies against IL-12 and IL-23 are described by Lacy et al. in WO 07/005,608. Recently, fully human antibodies derived from display libraries derived from human sources (Winter et al. U.S. Pat. No. 7,306,907; MacCafferty et al. U.S. Pat. No. 5,969,108) or from mice with human immunoglobulin transgenes have been described (Tomizuka et al. U.S. Pat. No. 7,041,870; Kucherlapati et al. U.S. Pat. No. 5,939,598). Salfeld et al. (U.S. Pat. No. 6,9141,28) describe fully human antibodies against IL-12.
Five broad classes of antibodies might be anticipated on the basis of interactions with IL-12, IL-23, IL-12p40 and IL-12p80 (FIG. 1). The first class of antibodies are those that specifically interact with IL-12p40 present in IL-12 and IL-23, along with the IL-12p40 monomer and the IL-12p80 homodimer (FIG. 1.1). The second class of antibodies are those that specifically interact with IL-12p35 (as exemplified by antibody G161-566 in Devergne et al. 2001 Am J Pathol 159 1763-76; FIG. 1.2). The third class of antibodies are those that specifically interact with IL-12 but not with IL-23, IL-12p35, and IL-12p40 (as exemplified by antibody 20C2 in D'Andrea, Rengaraju et al. 1992 J Exp Med 176 1387-98; FIG. 1.3). The fourth class of antibodies are those that specifically interact with IL-23p19 (as exemplified by Presta et al. WO 2007/027714; FIG. 1.4). The fifth class of antibodies are those that specifically interact with IL-23p40 but not with IL-12p40, exploiting sequence on the IL-12p40 subunit that is exposed on IL-23 but masked by the IL-12p35 subunit in IL-12 (as exemplified by Benson et al. U.S. Pat. No. 7,247,711; FIG. 1.5). This current invention describes novel forms of the first class of antibodies that specifically interact with IL-12p40, IL-12p80, IL-12 and IL-23 (FIG. 1.1).
Some information is known about the method by which this first class of antibodies inhibit the IL-12/23 receptor-ligand complex and thereby exert their antagonistic effect. Giles-Komar et al (WO 2006/069036) describe an anti-IL-12p40 antibody that is specific for amino acid residues 1-88 of IL-12p40. This antibody was further characterised as specifically inhibiting the interaction of IL-12 and IL-23 with IL-12Rβ1 (Papp et al. 2008 Lancet 371 1675-84). Likewise another antibody has been described in the literature as inhibiting the IL-12/23 interaction with IL-12Rβ1 (Ding et al. 2008 Curr Opin Investig Drugs 9 515-22). The present invention describes antibodies that inhibit the IL-12/23 receptor-ligand complex via a novel mechanism of action (FIG. 2.3). These novel mechanism of action involves the selective neutralisation of the IL-12/IL-12Rβ2 interaction and the IL-23/IL-23R interaction. They differ from antibodies described previously (see above) in that they do not neutralise the binding of IL-12/23 to IL-12Rβ1. These antibodies are also novel in that they do not inhibit IL-12p40/p80 binding to IL-12Rβ1 and thus do not inhibit the role IL-12p40/80 plays in host defense, thereby potentially increasing the safety profile of these antibodies relative to other IL-12/23 antibodies. Such antibodies with novel mechanisms of actions could lead to improvement in the treatment of diseases associated with IL-12/23 but with reduced safety concerns. Additionally, these antibodies could have improved efficacy since they do not inhibit the natural antagonists of IL-12 and IL-23, IL-12p40 or IL-12p80. This would allow both the antibody and IL-12p40 or IL-12p80 to function as antagonists thereby increasing the level of inhibition above that of administering the antibody alone.